Stem cells are a cell population possessing the capacities to self-renew indefinitely and to differentiate in multiple cell or tissue types. Embryonic stem cells (from approximately days 3 to 5 after fertilisation) proliferate indefinitely and can differentiate spontaneously into all tissue types: they are thus termed pluripotent stem cells (reviewed, for example, in Smith, A. G. (2001) Annu. Rev. Cell. Dev. Biol. 17, 435-462). Adult stem cells, however, are more tissue-specific and may have less replicative capacity: they are thus termed multipotent stem cells (reviewed, for example, in Paul, G. et al. (2002) Drug Discov. Today 7, 295-302). The “plasticity” of embryonic and adult stem cells relies on their ability to trans-differentiate into tissues different from their origin and, perhaps, across embryonic germ layers.
The ability of stem cells to self-renew is critical to their function as reservoir of primitive undifferentiated cells. In contrast, most somatic cells have a limited capacity for self-renewal due to telomere shortening (reviewed, for example, in Dice, J. F. (1993) Physiol. Rev. 73, 149-159). Stem cell-based therapies thus have the potential to be useful for the treatment of a multitude of human and animal diseases.
Stem cells as well as stem/progenitor cells can be derived from different sources. The “multi-lineage” potential of embryonic and adult stem cells has been extensively characterized. Even though the potential of embryonic stem cells is enormous, their use implies many ethical problems. Therefore, non-embryonic stem cells derived from the bone marrow stroma, fat tissue, dermis and umbilical cord blood have been proposed as alternative sources. These cells can differentiate inter alia into chondrocytes, adipocytes, osteoblasts, myoblasts, cardiomyocytes, astrocytes, and tenocytes in vitro and undergo differentiation in vivo, making these stem cells—in general referred to as mesenchymal stem cells—promising candidates for mesodermal defect repair and disease management.
In clinical use, however, harvesting of such mesenchymal stem cells causes several problems. The collection of the cells is a mental and physical burden to the patient as a surgical procedure is required to obtain the cells (for example, the collection of bone marrow is an invasive technique performed with a biopsy needle that requires local or even general anesthesia). Furthermore, in many cases the number of stem cells extracted is rather low. More importantly, no epithelial cells are derived or differentiated from these cells. This prompted the search for other possible sources of stem cells.
Umbilical cord blood has been identified as a rich source of haematopoetic stem/progenitor cells. However, the existence of mesenchymal stem/progenitor cells is discussed controversially. On the one hand, such cells could not be isolated or successfully cultured from term umbilical cord blood (Mareschi, K. et al. (2001) Haematologica 86, 1099-1100). At the same time, results obtained by Campagnoli, C. et al. (Blood (2001) 98, 2396-2402) as well as Erices, A. et al. (Br. J. Haematol. (2000) 109, 235-242) suggest that mesenchymal stem cells are present in several fetal organs and circulate in the blood of pre-term fetuses simultaneously with hematopoietic precursors. Accordingly, International Patent Application WO 03/070922 discloses isolation and culture-expansion methods of mesenchymal stem/progenitor cells from umbilical cord blood and a differentiation method of such cells into various mesenchymal tissues. Isolation efficiencies of about 60% have been reported (Bieback, K. et al. (2004) Stem Cells 22, 625-634). In the same study, both the time period from collection of the umbilical cord blood to isolation of the cells and the volume of the blood sample used have been determined as crucial parameters for achieving such a yield. However, it is still a matter of debate whether these stem/progenitor cells are indeed derived of umbilical cord tissue.
Recently, mesenchymal stem/progenitor cells have been successfully isolated from umbilical cord tissue, namely from Wharton's jelly, the matrix of umbilical cord, (Mitchell, K. E. et al. (2003) Stem Cells 21, 50-60; U.S. Pat. No. 5,919,702; US Patent Application 2004/0136967). These cells have been shown to have the capacity to differentiate, for example, into a neuronal phenotype and into cartilage tissue, respectively. Furthermore, mesenchymal stem/progenitor cells have also been isolated from the endothelium and the subendothelial layer of the umbilical cord vein, one of the three vessels (two arteries, one vein) found within the umbilical cord (Romanov, Y. A. et al. (2003) Stem Cells 21, 105-110; Covas, D. T. et al. (2003) Braz. J. Med. Biol. Res. 36, 1179-1183).
However, none of these approaches employed thus far has, for example, resulted in the isolation or cultivation of epithelial stem/progenitor cells as a source for epithelial cell-based therapies such as skin resurfacing, liver repair, bladder tissue engineering and other engineered surface tissues. Skin resurfacing is an especially critical and much needed medical treatment, which still needs a lot of development as can be seen from the numbers available for example for the USA. In the USA alone, there are 100.000 hospital treated burns per year and 600.000 cases of surgical skin excision. The age related problem of non-healing dermal wounds is far larger, with 11 to 12 million patients being treated in the USA. For these pathologies Europe shows approximately the same numbers of patients.
The skin has three layers, the epidermis, the dermis and the fat layer, which all perform specific tasks. The epidermis is generated principally by keratinocytes of epithelial origin, whereas the dermis is populated by fibroblastic cells of mesenchymal origin. The epidermis is the thin, tough, top layer of skin. The outer portion of the epidermis, the stratum corneum, is water-proof and, when undamaged, prevents most bacteria, viruses, and other foreign substances from entering the body. The epidermis also protects the internal organs, muscles, nerves, and blood vessels against trauma. The epidermis also contains islet-cells, which are part of the skin's immune system. The dermis, the next layer of skin, is a thick layer of fibrous and elastic tissue (made mostly of the polymers collagen and fibrillin) that gives the skin its flexibility and strength. The dermis contains nerve endings, glands, hair follicles and blood vessels.
The damage of the epidermis or dermis or of both layers (full thickness wounds) by mechanical force, such as abrasion, surgical wounds associated with the excision of skin cancers, or a loss of skin due to burns or other wounds, such as chronic venous ulcers, requires often the substitution of the skin. Depending on the extent of the damage the substitution with patient's own skin (autografts) taken from non-affected parts of the body is sometimes insufficient due to the extensive loss of skin. Therefore, a need exists to develop substitutes for damaged skin either with help of autologous or allogeneic cells.
U.S. Pat. No. 6,479,875, for example, describes a skin substitute, which consists of a scaffold which incorporates dermis-forming cells consisting of mesenchymal stem cells. However, these mesenchymal stem cells, which are isolated from the bone marrow, are rare and typically 10-20 cc aspirates have to be harvested from a patient in order to obtain enough mesenchymal stem cells.
Thus, there is still a need for methods and reliable sources useful for the isolation and cultivation of epithelial stem/progenitor cells, which can be used for the further development of adequate skin substitutes. Furthermore, rapid and efficient methods which are ethically acceptable and do not pose a biomedical burden on the patient for the isolation of epithelial and mesenchymal stem/progenitor cells are still required in order to provide such cells in a sufficient amount for various applications in regenerative medicine and tissue engineering.